In the earlier parent patent application the cloning and expression of a gene that encodes the bacteriophage T3 RNA polymerase, and the use of this RNA polymerase in making synthetic RNA probes were described. This transcription system requires plasmids that carry promoters that are recognized by the T3 RNA polymerase. The most useful type of transcription vectors carry both a T3 promoter and a promoter that is recognized by another phage RNA polymerase, usually that of bacteriophage T7 or bacteriophage SP6. In the earlier patent application the construction of a dual T3/T7 promoter vector designated pJFK9 was described and it was shown that under the conditions specified in the disclosure, that efficient transcription was initiated from the T3 promoter sequence. During subsequent testing of this plasmid, the results of certain experiments suggested that under very specific reaction conditions, the T3 promoter that had been cloned into pJFK9 functioned inefficiently. Specifically, when the concentration of one of the ribonucleoside triphosphates (uridine triphosphate) was lowered to 10 micromolar or less, initiation by the T3 RNA polymerase at the T3 promoter sequence occurred at low levels or was aborted. In reviewing the construction and sequence of pJFK9 it was noted that the sequence of nucleotides that lies downstream from the promoter site contained a thymidine residue at position +7 in the non-transcribed strand. From an initial sequence comparison of all the characterized, naturally occurring T3 promoters it was not obvious that the nucleotide at position +7 played an important role in the initiation of transcription. However, it was subsequently noted that thymidine had not been observed at this location in any of the naturally occurring T3 promoters thus far characterized. (Bailey, J. N. et al, Proc. Nat. Acad. Sci. USA 80:2814-2818 1983, which is incorporated herein by reference). Therefore, it was suspected that the unusual performance of the T3 promoter cloned into pJFK9 might have resulted from this base substitution in the downstream region. To circumvent this problem and to test this hypothesis, a new T3 promoter was synthesized that did not contain this particular change and which had a sequence in the downstream region that corresponded to one of the naturally occurring T3 promoters from positions -17 to +9 that had been previously characterized in the plasmid pJB30 (Bailey, J. N. et al, cited supra). Plasmid pJB30 carries a Taq I restriction fragment containing the strong T3 promoter located at 51.5 T3 map units.
This new synthetic T3 promoter was cloned into the plasmid pSP64 (which also contains a promoter for the bacteriophage SP6 RNA polymerase) resulting in the formation of a dual SP6/T3 promoter plasmid, which is designated pJFK15. The synthetic promoter in pJFK15 was subsequently removed and cloned into the plasmid pJFK9 in place of the synthetic T3 promoter which had previously been present in this plasmid. The new plasmid, which carries both synthetic T7 and T3 promoters, is designated pJFK16.
When the properties of pJFK16 were tested in vitro, it was found that this plasmid had the desired characteristics and performed well even at low concentrations of UTP.